Studies on Adsorption Kinetics and Thermodynamics of Macroporous Resin for Rosmarinic Acid.

This experiment treated perilla seeds with different concentrations of NaCl solution to enrich and purify their rosmarinic acid (RosA). The results showed that low concentrations of salt (0-20 mmol/L) promoted seed germination, while high concentrations (> 20 mmol/L) inhibited germination. When the salt concentration was 20 mmol/L, the germination rate was the highest. The content of RosA in germinated perilla seeds was 3.5 mg/g, which was 3.5 times as much as that in the seeds without germination. The RosA was purified using NK-109 macroporous resin and its adsorption kinetics, isotherms and thermodynamics were determined. The adsorption kinetics showed that the adsorption behavior of RosA in NK-109 resin conformed to the pseudo-second-order kinetic model. The model for RosA in the NK-109 resin exhibited Langmuir adsorption based on a spontaneous exothermic process according to its adsorption thermodynamics, which included both physical and chemical adsorption. The optimized process conditions were as follows: the loading concentration of 0.04 mg/mL, loading volume of 40 mL, 70% methanol as the eluent with the volume of 60 mL, and the purity of RosA was 42.1%.

Escherichia coli DNA polymerase I can disrupt G-quadruplex structures during DNA replication.

Non-canonical four-stranded G-quadruplex (G4) DNA structures can form in G-rich sequences that are widely distributed throughout the genome. The presence of G4 structures can impair DNA replication by hindering the progress of replicative polymerases (Pols), and failure to resolve these structures can lead to genetic instability. In the present study, we combined different approaches to address the question of whether and how Escherichia coli Pol I resolves G4 obstacles during DNA replication and/or repair. We found that E. coli Pol I-catalyzed DNA synthesis could be arrested by G4 structures at low protein concentrations and the degree of inhibition was strongly dependent on the stability of the G4 structures. Interestingly, at high protein concentrations, E. coli Pol I was able to overcome some kinds of G4 obstacles without the involvement of other molecules and could achieve complete replication of G4 DNA. Mechanistic studies suggested that multiple Pol I proteins might be implicated in G4 unfolding, and the disruption of G4 structures requires energy derived from dNTP hydrolysis. The present work not only reveals an unrealized function of E. coli Pol I, but also presents a possible mechanism by which G4 structures can be resolved during DNA replication and/or repair in E. coli.

A helical bundle in the N-terminal domain of the BLM helicase mediates dimer and potentially hexamer formation.

Helicases play a critical role in processes such as replication or recombination by unwinding double-stranded DNA; mutations of these genes can therefore have devastating biological consequences. In humans, mutations in genes of three members of the RecQ family helicases (blm, wrn, and recq4) give rise to three strikingly distinctive clinical phenotypes: Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome, respectively. However, the molecular basis for these varying phenotypic outcomes is unclear, in part because a full mechanistic description of helicase activity is lacking. Because the helicase core domains are highly conserved, it has been postulated that functional differences among family members might be explained by significant differences in the N-terminal domains, but these domains are poorly characterized. To help fill this gap, we now describe bioinformatics, biochemical, and structural data for three vertebrate BLM proteins. We pair high resolution crystal structures with SAXS analysis to describe an internal, highly conserved sequence we term the dimerization helical bundle in N-terminal domain (DHBN). We show that, despite the N-terminal domain being loosely structured and potentially lacking a defined three-dimensional structure in general, the DHBN exists as a dimeric structure required for higher order oligomer assembly. Interestingly, the unwinding amplitude and rate decrease as BLM is assembled from dimer into hexamer, and also, the stable DHBN dimer can be dissociated upon ATP hydrolysis. Thus, the structural and biochemical characterizations of N-terminal domains will provide new insights into how the N-terminal domain affects the structural and functional organization of the full BLM molecule.

[Structure and evolution of the eukaryotic FANCJ-like proteins].

The FANCJ-like protein family is a class of ATP-dependent helicases that can catalytically unwind duplex DNA along the 5'-3' direction. It is involved in the processes of DNA damage repair, homologous recombination and G-quadruplex DNA unwinding, and plays a critical role in maintaining genome integrity. In this study, we systemically analyzed FNACJ-like proteins from 47 eukaryotic species and discussed their sequences diversity, origin and evolution, motif organization patterns and spatial structure differences. Four members of FNACJ-like proteins, including XPD, CHL1, RTEL1 and FANCJ, were found in eukaryotes, but some of them were seriously deficient in most fungi and some insects. For example, the Zygomycota fungi lost RTEL1, Basidiomycota and Ascomycota fungi lost RTEL1 and FANCJ, and Diptera insect lost FANCJ. FANCJ-like proteins contain canonical motor domains HD1 and HD2, and the HD1 domain further integrates with three unique domains Fe-S, Arch and Extra-D. Fe-S and Arch domains are relatively conservative in all members of the family, but the Extra-D domain is lost in XPD and differs from one another in rest members. There are 7, 10 and 2 specific motifs found from the three unique domains respectively, while 5 and 12 specific motifs are found from HD1 and HD2 domains except the conserved motifs reported previously. By analyzing the arrangement pattern of these specific motifs, we found that RTEL1 and FANCJ are more closer and share two specific motifs Vb2 and Vc in HD2 domain, which are likely related with their G-quadruplex DNA unwinding activity. The evidence of evolution showed that FACNJ-like proteins were originated from a helicase, which has a HD1 domain inserted by extra Fe-S domain and Arch domain. By three continuous gene duplication events and followed specialization, eukaryotes finally possessed the current four members of FANCJ-like proteins.